Development of electron spin echo envelope modulation spectroscopy to probe the secondary structure of recombinant membrane proteins in a lipid bilayer

Abstract

Membrane proteins conduct many important biological functions essential to the survival of organisms. However, due to their inherent hydrophobic nature, it is very difficult to obtain structural information on membrane-bound proteins using traditional biophysical techniques. We are developing a new approach to probe the secondary structure of membrane proteins using the pulsed EPR technique of Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy. This method has been successfully applied to model peptides made synthetically. However, in order for this ESEEM technique to be widely applicable to larger membrane protein systems with no size limitations, protein samples with deuterated residues need to be prepared via protein expression methods. For the first time, this study shows that the ESEEM approach can be used to probe the local secondary structure of a 2 H-labeled d 8 -Val overexpressed membrane protein in a membrane mimetic environment. The membrane-bound human KCNE1 protein was used with a known solution NMR structure to demonstrate the applicability of this methodology. Three different a-helical regions of KCNE1 were probed: the extracellular domain (Val21), transmembrane domain (Val50), and cytoplasmic domain (Val95). These results indicated a-helical structures in all three segments, consistent with the micelle structure of KCNE1. Furthermore, KCNE1 was incorporated into a lipid bilayer and the secondary structure of the transmembrane domain (Val50) was shown to be a-helical in a more native-like environment. This study extends the application of this ESEEM approach to much larger membrane protein systems that are difficult to study with X-ray crystallography and/or NMR spectroscopy.

Electron Paramagnetic Resonance as a Tool for Studying Membrane Proteins

“…ESEEM spectroscopy is a pulsed EPR technique that is sensitive to systems containing weak dipolar couplings between an electron spin and a NMR-active nuclear spin. It can provide great insight into the structure and function of many important biological systems [91][92][93][94][95][96][97][98][99][100][101][102][103][104][105][106]. This ESEEM technique can measure a distance between a spin label and a single 2 H nucleus up to ~8 Å [107].…”

“…This ESEEM technique can measure a distance between a spin label and a single 2 H nucleus up to ~8 Å [107]. Nitroxide-based site-directed spin labeling ESEEM is very useful for probing the local secondary structure of membrane proteins/peptides in different environments including aqueous and lipid membranes [96][97][98][99][100][101][102][103][104][105]. The local secondary structure of membrane proteins has a great influence in the assembly, packing, and interaction of membrane proteins with their lipid membrane environment and hence is useful for understanding the function, dynamics, and interacting mode of membrane proteins [108,109].…”

“…These unique patterns provide pertinent local secondary structural information on α-helical structural motifs for protein/peptides using this ESEEM spectroscopic approach with short data acquisition times (~30 min) and small sample concentrations (~100 µM). This ESSEM approach has been applied to several biologically important protein/peptide systems such as the acetylcholine receptor (AChR) M2δ peptide, ubiquitin peptide, amphipathic model peptide LRL 8 , intermediate filament protein human vimentin, and KCNE1 to probe their local secondary structures [96][97][98][99][100][101][102][103][104][105]. Figure 7 membrane proteins [108,109].…”

Tension mediated mechanical activation and pocket delipidation lead to an analogous MscL state

“…The former involves the exchange of protons on a protein with deuterium generates accessibility information and has been used to provide key insights into the role of lipids in determining the conformational state, but also secondary structure changes in membrane transporters (40)(41)(42)(43)(44). The latter measures weak hyperfine coupling between unpaired electrons and nuclear spins to probe accessibility to solvent (D2O) and protons (residues, ions and lipids) and has been used to investigate the dynamics of membrane proteins (2,15,(45)(46)(47)(48)(49)(50)(51).…”

Utilization of 13C-labeled amino acids to probe the α-helical local secondary structure of a membrane peptide using electron spin echo envelope modulation (ESEEM) spectroscopy

“…Site-specific secondary structural information provides a better understanding of the functions, dynamics, and protein-lipid interactions of membrane proteins [4,14]. Not only has this ESEEM approach been successful for model peptides, it has also been utilized in over-expression systems to probe α-helical secondary structural motifs in both a water-soluble protein and a membrane protein, demonstrating the versatility of this technique [15,16].…”